Leucine and spermidine enhance shoot differentiation in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

Enhanced numbers of multiple shoots were induced from shoot tip explants of cucumber. The effects of amino acids (leucine, isoleucine, methionine, threonine, and tryptophan) and polyamines (spermidine, spermine, and putrescine) along with benzyladenine (BA) on multiple shoot induction were investigated. A Murashige and Skoog (MS) medium containing a combination of BA (4.44 μM), leucine (88 μM), and spermidine (68 μM) induced the maximum number of shoots (36.6 shoots per explant) compared to BA (4.44 μM) alone or BA (4.44 μM) with leucine (88 μM). The regenerated shoots were elongated on the same medium. Elongated shoots were transferred to the MS medium fortified with BA (4.44 μM), leucine (88 μM), and putrescine (62 μM) for root induction. Rooted plants were hardened and successfully established in soil with a 90% survival rate.     

Rapid In Vitro Propagation of East Indian Rosewood (Dalbergia latifolia Roxb.) through High Frequency Shoot Proliferation from Cotyledonary Nodes

An efficient protocol is described for large scale in vitro propagation of east Indian rosewood (Dalbergia latifolia Roxb.) using cotyledonary nodes derived from axenic seedlings. Of the four different cytokinins tested, BA was most effective in inducing multiple shoot buds in the explants. High frequency shoot proliferation (93%) coupled with maximum number of shoot formation (10-12 shoots/explant) was recorded on Murashige and Skoog’s medium supplemented with 2.0 mg/l BA. The frequency of shoot proliferation declined at higher levels of cytokinins. Shoot culture was multiplied by subculturing the original cotyledonary node on a fresh shoot multiplication medium each time aHer excising the newly formed shoots. Shoots obtained from each passage were multiplied further as nodal explants and each node produced 3-4 shoots. In two months from a single cotyledonary node, about 90 shoots were obtained. Rooting was induced in 72% of the regenerated shoots on half-strength MS containing IAA, IBA and IPA each at 1.0 mg/l. Rooted plantlets were hardened off and eventually established in soil.     

Differential cytokinin effects on the stimulation of in vitro shoot proliferation from meristematic explants of castor (Ricinus communis L.)

A highly efficient and reproducible method of in vitro propagation using meristematic explants has been developed for castor. Embryo axes and shoot tips were cultured on Murashige and Skoog (MS) medium supplemented with 0.5–10.0 mg/l of adenine, N⁶-benzyladenine (BA), kinetin (Kn), thiadiazuron (TDZ) and zeatin. TDZ (1.0–10.0 mg/l) gave the maximum number of shoots (37.8–40.0) from embryo axes, while BA (2.0 mg/l) was found superior to other cytokinins for obtaining the highest number of shoots (46.7) from the shoot apex. Adenine and Kn at all of the tested concentrations resulted in low proliferation rates from embryo axes. The carryover effect of the cytokinins was tested by subculturing proliferating shoot cultures from various media onto the medium fortified with 0.5 mg/l BA. There was no significant influence of the cytokinins on subsequent proliferation from the two explant types except for TDZ with embryo axes. The number of shoots from TDZ-habituated embryo axes ranged between 36.0 and 81.7, while it varied from 5.7 to 22.0 and 3.7 to 28.3 in axillary buds and embryo axes, respectively, on the other media. For elongation of shoots, gibberellic acid (GA₃) (0.1–1.0 mg/l) was added to the medium supplemented with 0.2–0.5 mg/l BA. Incorporation of GA₃ (0.1 mg/l) significantly enhanced the frequency of elongated shoots but drastically reduced the multiplication ability. Hence, proliferating shoot clusters were periodically transferred to the medium supplemented with 0.5 and 0.2 mg/l BA for further multiplication and elongation. Well-developed shoots were rooted on half-strength MS medium supplemented with 1.0 mg/l indole-3-butyric acid. The rooted plantlets were acclimatized with more than 60% success. Received: 17 June 1997 / Revision received: 3 September 1997 / Accepted: 20 September 1997     

High frequency shoot regeneration from nodal and shoot tip explants in Holarrhena antidysenterica L

Shoot tip and nodal segment explants of Holarrhena antidysenterica when cultured on MS medium containing BAP (1.0-3.0 mg/l) with NAA (0.2-1.0 mg/l) and BAP (1.0-3.0 mg/l) with Kn. (0.2-1.0 mg/l) produced multiple shoots. Maximum multiple shoots was found in MS medium supplemented with BAP (2.0 mg/l) and NAA (0.5 mg/l). Subculture on the same medium resulted in rapid shoot multiplication at an average rate of 16 new shoots per subculture. Addition of urea (100 mg/l) in the medium increased the number of shoots up to 22 per culture. For best rooting, the shoots were excised from the culture flask and implanted individually on half strength MS medium with 0.5 mg/l each of IBA, IAA and NAA. After 20 days of transfer on root induction medium 95% rooting was achieved. Regenerated plantlets were successfully acclimatized and established in soil. About 90% of plantlets survived under open field conditions.     

Direct shoot organogenesis and assessment of genetic stability in regenerants of <Emphasis Type="Italic">Solanum aculeatissimum</Emphasis> Jacq.

A simple and efficient procedure was developed for in vitro propagation of Solanum aculeatissimum Jacq. using leaf and petiole explants cultured on Murashige and Skoog (MS) medium supplemented with α-naphthalene acetic acid (NAA) and 6-benzyladenine (BA). Effects of various plant growth regulators, explant types, carbohydrates, and basal salts on induction of adventitious shoots were also studied. Leaf explants appeared to have better regeneration capacity than petiole explants in the tested media. The highest regeneration frequency (79.33 ± 3.60%) and shoot number (11.33 ± 2.21 shoots per explant) were obtained in leaf explants in MS medium containing 3% sucrose and 0.8% agar, supplemented with 0.1 mg/l NAA and 2.0 mg/l BA, whereas petiole explants were more responsive to 0.1 mg/l NAA and 1.0 mg/l thiadiazuron. Developed shoots rooted best on MS medium with 1.0 mg/l indole acetic acid (IAA), producing 18.33 ± 2.51 roots per shoot. Histological investigation showed that the shoot buds originated mainly from epidermal cells of wounded tissues, without callus formation. The regenerated plantlets were successfully acclimatized in a greenhouse, where over 90% developed into morphologically normal and fertile plants. Results of flow cytometry analysis on S. aculeatissimum indicated no variation in the ploidy levels of plants regenerated via direct shoot formation and showed almost the same phenotype as that of mother plants. This adventitious shoot regeneration method may be used for large-scale shoot propagation and genetic engineering studies of S. aculeatissimum.     

A rapid in vitro propagation of red sanders (<Emphasis Type="Italic">Pterocarpus santalinus</Emphasis> L.) using shoot tip explants

An efficient protocol has been developed for the in vitro propagation of Pterocarpus santalinus L. using shoot tip explants which is a valuable woody medicinal plant. Various parts of this plant are pharmaceutically used for the treatment of different diseases. Multiple shoots were induced from shoot tip explants derived from 20 days old in vivo germinated seedlings on 1:1 ratio of sand and soil after treating with gibberellic acid (GA₃). The highest frequency for shoot regeneration (83.3%) with maximum number of shoot buds (11) per explant was obtained on Murashige and Skoog (MS) medium supplemented with 1.0 mg/l of 6-benzylaminopurine (BAP) along with 0.1 mg/l of thidiazuron (TDZ) after 45 days of culture. A proliferating shoot culture was established by repeatedly subculturing the original shoot tip explants on fresh medium after each harvest of the newly formed shoots. Sixty percent of the shoots produced roots were transferred to rooting medium containing MS salts and 0.1 mg/l indole-3-butyric acid (IBA) after 30 days. About 73.33% of the in vitro raised plantlets were established successfully in earthen pots. Random amplified polymorphic DNA (RAPD)-based DNA fingerprinting profiles were generated for the first time using shoot tip explants of this species and confirmed that there was no genetic variability. This protocol might be helpful for the mass multiplication of P. santalinus in the future.     

Rapid in vitro multiplication of Drosera indica L.: a vulnerable, medicinally important insectivorous plant

An efficient protocol is described for rapid in vitro multiplication of the vulnerable medicinal herb Drosera indica L. by enhanced axillary bud proliferation from shoot tips as explants. In order to standardize in vitro multiplication of D. indica, the effects of different strengths of Murashige and Skoog (MS) medium (1/4, 1/3, 1/2 and full strength), different percentages of sucrose (1, 2 and 3%), various pH (3.7, 4.7, 5.7 and 6.7) and MS basal medium fortified with different concentrations of zeatin (Z), kinetin (KN) (0.1, 0.5, 1.0 and 2.0 mg/l) and 6-benzylaminopurine (BA) (0.01, 0.05 and 0.1 mg/l) were tried. Multiple shoot production was independent of different strengths of MS, various percentages of sucrose and also when pH was altered. Although the number of multiple shoots developed on MS medium supplemented with Z (0.1, 0.5, 1.0 and 2.0 mg/l), KN (0.5 and 1.0 mg/l) and BA (0.1 mg/l) separately was high, the maximum number was observed on MS fortified with Z (0.5 mg/l) and KN (0.5 mg/l), respectively, which clearly depicts that there is not much difference comparatively with a variation in hormone concentration in case of Z. High cytokinin concentrations resulted in retardation of shoot growth. Rooting was best achieved on MS basal medium. This protocol could be useful for production of large biomass within 6 weeks for plumbagin bioprospection and long term in vitro conservation.     

香蕉组织培养过程中内生菌污染的控制

研究了体外培养一种孟加拉传统香蕉(Musa spp.Cv. Kanthali)的茎尖组织。茎尖的原始细胞表面经无菌处理(0.1%HgCl2处理12min) ,接种6~15d后外植体地下茎部分仍有微生物污染(大部分是细菌) ,杀死了85%的外植体。为确定无污染培养基,将等量外植体分别浸泡在含400mg/L氨苄青霉素和200mg/L庆大霉素(两种光谱抗生素)的培养基中1h。结果表明,经抗生素处理的外植体完全没有污染,但培养3周后不能再生。进行二次继代培养后,其中一部分外植体吸收了培养基并胀大,颜色由苍白转变成浅绿或深绿。三次继代培养后数天,不再观察到外植体的生长,所有经抗生素处理过的外植体都开始死亡。在未经抗生素处理的活外植体中,单个茎发育的最佳培养基是:MS 4.0mg/L BA 0.5mg/L KT 15% CW,平均生长时间为18~21d,但再生率很低,只有30%。茎细胞增殖的最佳培养基是:MS 4.0mg/L BA 2.0mg/LIAA 15% CW,每个茎平均只萌发3~4个芽。最后,在添加0.5mg/LIBA的一半浓度的MS培养基中,体外培养茎最大生根率达到90%。     

In vitro propagation of emetic nut Randia dumetorum (Lamb.)

An efficient protocol for in vitro shoot multiplication of Randia dumetorum (Emetic nut) has been developed. The seeds of R. dumetorum were germinated in vitro in MS medium in 5 weeks. Subsequent propagation using shoot tip as an explant was carried out in MS medium along with different concentrations and combinations of BAP (0.5-2.0) and NAA (0.0-2.0). Maximum shoot multiplication was obtained (12.7 shoots per shoot tip) in MS medium containing 1 mg/L BAP and 1 mg/L NAA. Micropropagated shoots were rooted in 1/2 MS medium supplemented with 1 mg/l IBA. This is the first report of in vitro plant propagation of R. dumetorum. In vitro grown plantlets showed a survival rate of 70% after 2 months of transplantation to natural environment.     

AgNO3 boosted high-frequency shoot regeneration in Vigna mungo (L.) Hepper

In order to further increase shoot regeneration frequency of Vigna mungo (L.) Hepper., the effects of AgNO₃ on this process was investigated in this study. The shoot tip and cotyledonary node explants were cultured on MS salts B5 Vitamins medium containing BA+TDZ+Ads+AgNO₃ for multiple shoot induction. AgNO₃ influenced the shoot bud formation and their subsequent proliferation. The best medium composition for multiple shoot induction was BA, TDZ combination with Ads and AgNO₃ in MSB5 medium. Maximum 39 shoots in cotyledonary node and 22 shoots in shoot tip were obtained per explants after 4 – 6 wk. of culture. Elongation and rooting were performed in GA₃ (0.6mg/l) and IBA (0.4mg/L) containing media respectively. The in vitro raised plantlets were acclimatized in green house and successfully transplanted to the field with a survival rate of 78%.     

Rapid in vitro multiplication and ex vitro rooting of Rotula aquatica Lour., a rare rhoeophytic woody medicinal plant

Single medium-based efficient protocols for large-scale multiplication of the rare woody aromatic medicinal plant Rotula aquatica Lour. by means of axillary bud multiplication and indirect organogenesis were established using Murashige and Skoog (MS) medium. There were no significant differences with respect to the induction of shoots per node or callus and roots per shoot on media prepared either with tap water and commercial sugar or those prepared with double distilled water and tissue culture-grade sucrose. The most effective medium for axillary bud proliferation was MS medium fortified with 1.0 mg l(-1 )N(6)-benzylaminopurine (BAP) and 0.5 mg l(-1 )indole-3-butyric acid (IBA), on which shoots were induced at the rate of 15 per node. The excision of node segments from the in vitro-derived shoots and their subsequent culture on medium supplemented with same concentrations of BAP and IBA facilitated enhanced axillary bud proliferation. Callus that developed from the lower cut end of the node explants induced shoots during subculture on half-strength MS medium with 1.0 mg l(-1 )BAP and 0.5 mg l(-1 )kinetin. The shoots developed rooted best on half-strength MS medium supplemented with 0.5 mg l(-1 )naphthaleneacetic acid (NAA). Rooted shoots, following acclimation in the greenhouse, were successfully transferred to field conditions, and 80% of the plantlets survived. When the basal ends of shoots harvested from multiplication medium were dipped in an NAA (0.5 mg l(-1)) solution for 25 days, a mean of 5.6 roots per shoot developed; the transfer to small pots facilitated the survival of 75% of the rooted shoots. Ex vitro rooting by direct transfer of the shoots from the multiplication medium to the greenhouse resulted in a 65% survival. Commercial sugar and tap water and ex vitro rooting make the protocol economically advantageous. About 750 plantlets were procured in a 3-month period starting from a single node explant.     

Artemisinin production by shoot regeneration of Artemisia annua L. using thidiazuron

An efficient in vitro method for multiple shoot bud induction and regeneration has been developed in Artemisia annua L. using leaf and stem explants in various concentrations and combinations of plant growth regulators to evaluate the frequency of regeneration. The sources of explants as well as plant growth regulators in the medium were found to influence the multiple shoot induction. The result shows that the stem segment cultured on Murashige and Skoog (MS) medium supplemented with 0.1 mg/l thidiazuron (TDZ) gave a perfect shoot formation (100%) and good shoot multiplication (57 shoots/explant) after 2 weeks of culture. Healthy regenerated shoots were elongated and rooted in MS medium without hormones. The artemisinin content in plants regenerated from stem explants using 0.1 mg/l TDZ was (3.36 +/- 0.36) microg/mg dry weight and two-fold higher than that of in vitro grown plants of the same age [(1.73 -/+ 0.23) microg/mg DW]. This system exhibited a potential for a rapid propagation of shoots from the stem explant and makes it possible to develop a clonal propagation of A. annua.     

Reduction of vitrification in in vitro raised shoots of Chlorophytum borivilianum Sant. & Fernand., a rare potent medicinal herb

Reduction of vitrification in in vitro raised shoots derived from shoot bases and immature floral buds along with inflorescence axis used as explants of C. borivilianum, a rare medicinal herb is described. Shoot multiplication was obtained on MS medium with 2 mg l(-1) benzylaminopurine (BAP) + 0.1 mg l(-1) indole-3-butyric acid (IBA) and MS medium with 2 mg l(-1) kinetin (Kin) + 0.1 mg l(-1) 2,4-dichlorophenoxy acetic acid (2,4-D) from shoot bases and inflorescence axis respectively. Best multiplication rates were obtained from both the explants on MS medium with 2 mg l(-1) BAP. Vitrification of shoots in cultures appeared during the multiplication stage. Culture bottles with aerated caps reduced the vitrification to 80%. Reduction of BAP concentration from 2 mg l(-1) to zero during subsequent subcultures also minimized vitrification. Use of 0.5-2 mg l(-1) Kin produced healthy shoots when compared to BAP. In vitro raised shoots rooted on Knop salts containing iron and vitamins of MS medium, 2 mg l(-1) IBA and 0.1% activated charcoal. About 80% plantlets survived upon soil transfer. Scanning electron microscopic and image analyzer studies reveal the morphological structural differences between the leaves of normal and vitrified plantlets.     

Efficient clonal propagation method for Decalepis hamiltonii, an endangered shrub, under the influence of phloroglucinol

A highly efficient two stage protocol was developed for induction of multiple shoots from single node in vitro shoot tip explants of Decalepis hamiltonii. It was found that phloroglucinol (PG) had synergistic effect on shoot multiplication when added with N6-benzyladenine and gibberellic acid. This protocol uses PG for both multiple shoot induction from nodal explants, elongation of primary shoots and initiation of adventitious shoot formation from primary shoots, which was more in presence of triacontanol (TRIA). Maximum number of shoots per culture was observed on the medium containing N6-benzyladenine (1.1 microM; BA), GA3 (5.8 microM) and PG (800 microM). Sub-culturing of the shoots onto MS medium containing optimum concentration of BA (5.6 microM), PG (200 microM) and TRIA (0.011 microM) produced elongated shoots along with secondary shoot formation. The long shoots were rooted on alpha-naphthalene acetic acid (5.38 microM; NAA) and PG (400 microM) containing medium. The rooted plantlets were hardened and their field survival rate was 80-90%.     

Micropropagation of a fruit tree, Morus australis Poir. syn. M. acidosa Griff.

High frequency bud break and multiple shoots were induced in nodal explants collected between November to February from a 5 year old tree of Morus australis Poir syn. M. acidosa Griff. on Murashige and Skoog's medium supplemented with 6-benzylaminopurine (1.0 mg/1). Incorporation of gibberellic acid (0.3 mg/l) along with BAP (1.0 mg/l) not only induced faster bud break from nodal explants as well as from apical shoot buds, but it also enhanced the frequency of bud break. Nodal explants were more responsive than apical shoot buds. The shoots formed in vitro were multiplied further as nodal segments, and an average multiplication rate of 6-fold per subculture was established within 4–5 months. The shoots were successfully rooted on half-strength MS containing a combination of indole-3-acetic acid, indole-3-butyric acid and indole-3-propionic acid, each at 1.0 mg/1. The plantlets were successfully hardened off and established in natural soil.Abbreviations BAP 6-benzylaminopurine - GA₃ gibberellic acid - KN kinetin - IAA indole-3-acetic acid - IBA indole-3-butyric acid - IPA indole-3-propionic acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthalene acetic acid     

Micropropagation of Coleus blumei from nodal segments and shoot tips

A rapid and highly-effective method for micropropagation from nodal segment and shoot tip explants was established for Coleus blumei Benth. Nodal segments and shoot tips were inoculated on MS medium containing 0.7 % agar, 3 % commercial sugar, and different combinations of 6-benzyladenine (BA) with indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or α-naphthaleneacetic acid (NAA). Hundred percent shoot induction from both explants was achieved on the medium containing BA (2 mg dm⁻³) and NAA (1 mg dm⁻³). Shoot tips were proved to be the better explant in comparison to nodal segments in having high rate of shoot induction and more number of shoots. The same media conditions were found suitable for shoot multiplication. Multiplied shoots rooted best on MS medium supplemented with IBA (2 mg dm⁻³). Micropropagated plants were successfully established in soil after hardening, with 100 % survival rate.     

In vitro rapid multiplication and propagation of <Emphasis Type="Italic">Cardiospermum</Emphasis><Emphasis Type="Italic">halicacabum</Emphasis> L. through axillary bud culture

A simple, rapid and efficient protocol for micropropagation of Cardiospermum halicacabum via axillary bud multiplication has been successfully developed. The organogenic competence of nodal segments was investigated on Murashige and Skoog (MS) medium supplemented with different concentrations of benzyladenine (BA), kinetin (Kn), thidiazuron (TDZ) and 2-isopentenyladenine (2-iP). Multiple shoots differentiated directly without callus mediation within 4 weeks when explants were cultured on a medium fortified with cytokinins. The maximum number of shoots (14.83 ± 0.52) was developed on a medium supplemented with 0.3 μM TDZ. Such proliferating shoots when subcultured onto MS media devoid of TDZ gave the highest rate of shoot multiplication (35.66 ± 1.00) by the end of fourth subculture passage. Elongated shoots were rooted on 1/3 MS medium augmented with 0.5 μM IAA. The plantlets thus obtained were successfully hardened and transferred to greenhouse.     

Mass propagation of <Emphasis Type="Italic">Plectranthus bourneae</Emphasis> Gamble through indirect organogenesis from leaf and internode explants

The present study describes the plant propagation via indirect organogenesis from in vitro derived leaf and internode explants of Plectranthus bourneae, an endemic plant to south India. Leaf and internodal explants successfully callused on Murashige and Skoog medium (MS) supplemented with different concentrations of auxins [2,4-D (2,4-dichlorophenoxyacetic acid), NAA (α-naphthalene acetic acid), IAA (indole-3 acetic acid), IBA (indole-3-butyric acid) and PIC (Picloram); 0.1–2.0 mg/l] in combination with BA (6-benzyladenine) (0.5 mg/l). Maximum callus induction (98 %) was achieved from leaf explant followed by internodal explant (89 %) at 1.0 mg/l NAA, 0.5 mg/l BA. Leaf derived callus showed better shoot regeneration (29.71 shoots) on MS medium containing 1.0 mg/l KN (kinetin), 0.7 mg/l NAA, and 50 mg/l CH (casein hydrolysate) followed by internodal callus (19.71). A maximum of 19.14 roots/shoot was observed at 1.0 mg/l IBA. The rooted plantlets were successfully hardened and transferred to greenhouse condition with 80 % survival. This system could be utilized for large-scale multiplication of P. bourneae by tissue culture.     

A protocol toward multiplication of the medicinal tree,Eucommia ulmoides oliver

Summary A protocol based on shoot cultures of 1-mo.-old seedlings was developed for rapid asexual multiplication of eucommia, the source of an antihypertensive medicinal. The explant is an excised shoot tip, 3–5 mm tall. MS basal medium supplemented with 1 mg/liter BA is employed to establish primary cultures and subsequently multiply shoots. Shoots are subculturable on the same medium and can be increased at a rate of 7.5 new shoots per 2-shoot sector every 3 wk. Rooting is achieved in a Gelrite medium with the MS salts reduced to 1/3 strength and the BA replaced by 0.1 mg/liter NAA. The method is not directly applicable to mature trees. Applicability will require explants from rejuvenated sources, possibly attainable by the method of repeated grafting of shoot apices onto juvenile rootstocks, repeated subculturing of shoots, or culturing shoot apical meristems.     

The Effect of Plant Growth Regulators and Sucrose on the Micropropagation and Microtuberization of <Emphasis Type="Italic">Dioscorea nipponica</Emphasis> Makino

The effects of sucrose, plant growth regulators, MS (Murashige and Skoog), and ½MS salt media formulations were investigated for the development of shoot cultures, microtuber induction, and plantlet regeneration in Dioscorea nipponica. The cytokinin N-benzyladenine (BA) in the range of 0.5–2.0 mg/l showed strong enhancing effects on microtuber induction only when used in conjunction with the auxin alpha-naphthalene acetic acid (NAA), with the effect that NAA increased from 0.5 to 2.0 mg/l. Murashige and Skoog salt media supplemented with sucrose at 3% (w/v) gave the highest frequencies of shoot induction (86%) when BA was present at 2.0 mg/l and NAA at 1.0 mg/l. Sucrose at 7% (w/v) was the single most significant medium constituent for microtuber growth. The heaviest microtubers were formed on media containing 1.0 mg/l BA and 2.0 mg/l (0.073 g), especially with 7% sucrose (3.46 g). With media containing ½MS, 2% sucrose, and 0.1% (w/v) activated charcoal, the percentage of rooting was maximal when supplemented with 1.0 mg/l BA and 0.5 mg/l NAA for the in vitro produced shoots (95%) and BA and NAA both at 0.5 mg/l for the microtubers (100%). When removed from culture flasks and transferred into sterilized soil in a greenhouse, most of the hardened plantlets survived (over 91% after 1 week), and they were suitable for field planting after 1 month.